The benefits of AQIX RS-I over Krebs & Henseleit in isolated mammalian preparations

Theory behind why AQIX® RS-I has advantages over Krebs & Henseleit
The components in the formulations of AQIX® RS-I and Krebs & Henseleit
Comparative cell necrosis as demonstrated by leakage of Lactate Dehydrogenase (LDH)
Comparative survival periods of animal and human isolated preparations
Comparative twitch response in skeletal muscle preparations
Comparative cell receptor response in human colon biopsies
Comparative HT receptor response in rabbit ciliary ganglion neurones
Improved survival and functional viability of human bronchial biopsies
Improved survival and functional viability of human atrial trabeculae preparations
Published negative effects of phosphate radicals on isolated tissues/organs

1. Theory behind why AQIX® RS-I has advantages over Krebs & Henseleit

In common with typically used perfusion solutions, Krebs & Henseleit is pH buffered with inorganic phosphate radicals at 1-2 mmole/L, at which concentration, they have been shown to gradually precipitate calcium and magnesium ions as insoluble phosphate salts. However, in the interstitial fluid surrounding all mammalian cell membranes, inorganic phosphate ions only occur in low concentrations (0.12 mmoles/L) whereby such precipitations do not occur.

Calcium ions need to be kept at a constant concentration – about l00,000 times lower inside the cell membrane compared to the outside where they facilitate the maintenance of the ADP:ATP energy cycle which is required to translocate calcium ions into the mitochondrial matrix to maintain homeostasis.

Magnesium ions are essential co-factors for the enzymatic conversion of glucose to glucose 6-phosphate and fructose to fructose 6-phosphate and many other enzymes comprising the Glycolytic Pathway (below). Inhibition of glycolysis prevents the formation of pyruvate, a 3-carbon amino acid and essential for entry into the Krebs cycle for the production of ATP. This probably explains why phosphate buffered salines contain pyruvate, added at unnatural serum concentrations (1-2 mmole/L) to by-pass the inhibitory effect of magnesium chelation within the Glycolytic cycle (see below). However, abnormal levels of pyruvate cause end-product inhibition within the Glycolytic cycle and strongly inhibit associated LDH-subunits, which as a consequence, prevents the supply of 3-carbon units is with cessation of ATP synthesis. Similarly, D-gluconate and citrate pH buffered solutions (e.g., Soltran®) prevent the entry of D-glucose and D-fructose into the glycolytic pathway by inhibiting the enzyme, hexokinase (HK).

In direct contrast, AQIX® RS-I maintains cellular homeostasis so enabling the free-flow of the glycolytic cycle to maximize substrate utilization by optimizing enzyme (HK/PFK) activity and ultimate ATP synthesis in Krebs cycle.

2. A comparison of the components of human serum, interstitial fluid, AQIX RS-I and Krebs & Henseleit

Cations (mOsmoles/L)	Serum [mean]	ISF	RS-I [2000]	K & H [1932]
Sodium	139.5	136	136	143
Potassium	4.3	4.4	5.0	5.9
Calcium (total)	2.5	1.18	b 1.25	2.5
Magnesium (total)	2.4	0.51	b 0.45	1.2
TOTAL	148.7	142.1	142.7	152.6
Anions (mOsmoles/L)	Serum [mean]	ISF	RS-I [2000]	K & H [1932]
Chloride	106	117	118	143
Bicarbonate	25	23.9	25	24.8
Phosphate (inorg.) [total]	1.0	0.61	0.0	1.2
Sulphate	0.5	0.5	0.0	1.2
Organic acids	6.4	7.0	5 (BES)	0.0
Proteins	2.0	1.0	0.0	0.0
Albumin	0.65	0.19	0.0	0.0
TOTAL	137.5	154.5	148	154.2
GLUCOSE	4.9	3.6	10.0	11.1
GLYCEROL	0.13	0.09	0.11	0.0
INSULIN (nmoles)	1.4	1.5	1.7	0.0
OSMOLALITY (mOsmoles/kg)	264-290	265-286	264-282	328
CONDUCTIVITY (mS cm-1)	11.7 – 12.3	11.9 – 12.6	11.8 – 12.2	N/A

3. Comparative cell necrosis as demonstrated by leakage of Lactate Dehydrogenase (LDH)

Leakage of Lactate dehydrogenase (LDH) in isolated rat liver preparations as is an indicator of cellular apoptosis and necrosis.

‡ In the comparison between AQIX® RS-I and K&H in the perfused rat liver, where only the hepatic portal vein was perfused, all 5 of the K & H preparations died after 150 minutes compared to the 100% survival at this time in AQIX® RS-I.

4. Comparative survival periods of isolated preparations

Functional viability under normothermic and mild hypothermic conditions.

Species	Tissue/Organ Preparations	Maxm Days Stored	Preservation in vitro °C	Conditions Exp. °C
rat	jejunum	9.0	8-12	35
	jejunum	1.5	-	35
	ileum	8.0	8-12	35
	ileum	1.3	-	20-35
	colon	5.0	-	20-35
	uterus	3.0	-	35
	uterus	10.0	8-12	35
	detrusor muscle	2.0	-	20-35
	diaphragm muscle	0.6	-	35-37
	diaphragm muscle	2.0	-	20-35
	soleus muscle	1.1	-	20-35
	heart	0.8	-	35-37
	heart	2.1	-	20-25
	heart-lung	1.2	-	20-35
	RBC’s	4.0	No haemolysis at 4°C
	kidney	1.0	-	20-35
	‡ liver	0.3	-	35
rabbit	intestine (jejunum)	5.0	8-12	37
	intestine (jejunum)	2.0	-	20-37
	uterus	7.0	8-12	37
	superior cervical	2.0	8-12	37
	ganglion	0.8	-	37
	RBC’s	3.0	No haemolysis at 4°C
guinea pig	ileum	7.0	8-12	37
	detrusor muscle	4.0	8-12	37
	detrusor muscle	1.0	-	20-37
	heart	0.4	-	20-37
mouse	soleus muscle	0.9	-	20-35
	diaphragm muscle	1.5	-	20-35
	intercostal muscle	0.9	-	20-35
pig	kidney	0.8	0-4	37
	kidney	0.8	30	37
human	intercostal muscle	1.3	-	37
	kidney (organ)	1.5	0-4	37
	intestine(organ)	0.3	-	37
	colon (biopsy)	1.3	0-4	-
	lung(organ)	0.3	-	37
	lung bronchi (biopsy)	0.8	-	37
	atrial trabeculae(biopsy)	0.6	-	37
	liver(organ)	0.3	-	37
	RBC’s	1.3	0-4	-
	RBC’s	0.3	-	37
	Leucocytes	0.8	-	37

Tabulation of the recorded periods (days) of maintained physiological/pharmacological viability of tissue/organ preparations from different mammalian species following isolation in AQIX® RS-I preservation/ perfusion solution. The various preparations depicted in this table were maintained under varying in vitro conditions, e.g., stored, perifused or simultaneously perfused/perifused at the temperatures indicated.

These observations reflect the versatility of AQIX® RS-I solution in accommodating the differing physiological and metabolic demands of tissue/organ preparations from a variety of mammalian species.

5. Comparison of twitch responses in rat skeletal muscle preparations

Twitch responses are approximately 50% lower in Krebs & Henseleit perfused tissue over time.

6. Comparative cell receptor response in human colon - an independent study

Biopsied Tissues 1 to 4 were procured and transported over ice in PBS and then perfused normothermically in Krebs & Henseleit.

Biopsied Tissues 5 to 8 were both procured and transported over ice in AQIX® RS-I and also perfused normothermically in AQIX® RS-I.

Both the magnitude and contours of the carbachol response curves are totally different. The tissue preparations in AQIX® RS-I are responding as they would clinically, and results from both the activation of cholinergic receptors on the visceral musculature and variation in potassium ion conductivity in unison with the continued on-going activity of intrinsic neurons. Neuronal activity in rat visceral musculature (uterus) has have been demonstrated to last only a few hours in Krebs buffered solutions compared to up to 10 days in AQIX® RS-I. Comparable results have been achieved using guinea pig ileum biopsies stored at 0 - 4°C for up to 7 days.

7. Comparative HT receptor response in rabbit ciliary ganglions

AQIX® RS-I and Krebs & Henseleit solutions as perfusion and preservation media for use in HT-receptor applications using rabbit ciliary ganglionic preparations.

Solution	Preparation Viability	Conditions
K & H	< 4 hours	@ 37.5 °C
AQIX® RS-I	> 10 hours	@ 37.5 °C

8. Improved survival and functional viability of biopsied human bronchial muscle biopsies

9. Improved survival and functional viability of biopsied human atrial trabeculae preparations

Methods:

Results:
Milrinone, as shown in Figures 2a & b, increased cardiac muscle contractility in a concentration-dependant manner over the range 1 x 10-8 M to 1 x 10-5 M. Figure 2a demonstrates that the positive inotropic effect of milrinone was reversed by the addition of a single high concentration (1 x 10-5 M) of the muscarinic agonist, acetylcholine (Ach).

10. Observed negative effects of phosphate radicals on isolated tissues/organs

CREESE (1950) - rat hemidiaphragm perifused with ; pCO2/HCO3 limited LOSS of K+ but GAIN of Na+ H2PO4-/HPO4- NEGATIVE inotropism NOT reversed with HCO3

STADIE & SHAW (1959) - rat hemidiaphragm perifused with ; H2PO4-/HCO3- - caused INHIBITION of HK & PFK enzymes

BERMAN & SAUNDERS (1955) - rat ventricle perfused with; D-glucose INEFFECTIVE substrate in PO4- - based media

SVEDMEYR (1965) - rat ventricle perfused with; glycogenolytic effect of epinephrine LESS in PO4- - based media than in HCO3—based media

KO & PARADISE (1969) - rat atria perfused with; ONLY HCO3 based media could RESTORE ATP levels

EAGLE (1971) - cell culture preparations incubated with; HCO3 based media INDISPENSABLE for CELL GROWTH

HALL & De LUCA (1986) - rat heart mitochondrial creatine kinase (CK) incubated with; PO4 based media caused IRREVERSIBLE INHIBITION of CK

De FRIETAS & VALENTINE (1984) - superoxide dismutase preparations incubated with; PO4 based media caused INHIBITION of SOD

STEWART ET AL. (1986) - sheep heart transplants preserved with; HCO3 based media containing SOD sustained left venticular function upon transplantation/reperfusion

REES & DALZEIL (1994) - isolated rat diaphragm perfused with; PO4 based Krebs & Henseleit perfusate showed 30% decline in twitch contractions by 90 minutes compared to 7% in RS-I solution. Proceedings of the Anaesthesiology Group of New Zealand, May, 1994, 4 pp.

If you would like to trial a sample of AQIX RS-I or require further information, please contact us. More information about the variety of applications for this patented, fluid technology can viewed by selecting a link below:

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Cell & Tissue Preservation

Transplant Perfusion

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